Preparing an implant by gas-plasma treatment of a substrate to couple cells

ABSTRACT

An implant for use in biological/biomedical applications may be prepared by subjecting a substrate to a gas-plasma treatment. The substrate may be a biocompatible material, including metals, ceramics, and polymers. More specifically, the substrate may be a bioresorbable polymer. The gas-plasma treatment may include subjecting the substrate to a plasma formed by a reactive gas. The gas-plasma treatment may be performed for a chosen duration at a radio frequency within a temperature range, a pressure range, and a supplied energy range. The substrate may be exposed to living cells, such that some of the living cells become coupled to the substrate. Gas-plasma treatment parameters may be chosen such that the living cells coupled to the treated substrate produce more of a cellular product than living cells coupled to an untreated substrate.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention generally relates to gas-plasma treatment of substrates.More specifically, the invention relates to preparing an implant for usein biological/biomedical applications by subjecting the substrate to agas-plasma treatment.

2. Description of Related Art

Successful repair or replacement of damaged or diseased tissues, organs,or bones requires integration of donor tissue or an implant with apatient's body. While tissue from the patient or from another donor maybe integrated more readily than an implant, tissue transplantation has ahigh rate of failure and is accompanied by a risk of diseasetransmission. In addition, donor tissue may be in short supply, andtransplantation procedures may be expensive. Implants used as medicalimplants have therefore been increasingly investigated as alternativesto donor tissue.

Although implants circumvent some of the problems associated with donortissue, the use of implants introduces other problems. An artificial orsynthetic implant may be incompatible with the body and may not functionas well as the original tissue. Since the surface properties of animplant generally govern the interaction of the implant with the body,rejection and other medical complications are often traced toincompatible implant surfaces. Inert implants, once thought to bedesirable, are now known to cause surrounding growth of fibrous tissue,thereby inhibiting integration of the implant with a patient's body.Biocompatible implants are believed to reduce this and other adversemembrane-mediated cellular responses, such as infection, inflammation,blood coagulation, etc., related to the presence of foreign matter inthe body. Thus, considerable attention has been directed towardincreasing biocompatibility of implants through modification of implantsurfaces to mimic functionally equivalent surfaces in the body.

Implants are typically made from metals, polymers, and ceramics. Thesematerials, however, do not generally adhere to host tissue or coatingsdesigned to enhance implant biocompatibility. Therefore, implantsurfaces are often conditioned or treated to increase adhesion to thehost tissue or to a desired coating composition. A purpose of suchtreatment is to enhance integration of an implant with adjacent hosttissue. Successful surface modification results in an implant withsurface characteristics that allow adhesion of a desired coating.Implants thus treated allow ingrowth, or integration of host tissue withthe implant coating.

A goal of forming biocompatible implants is choosing the most effectivetreatment and coating for the intended application. Coatings typicallyinclude inorganic, polymeric, and biological coatings. Since endothelialcells form blood-surface interfaces in the human body, the attachment ofendothelial cells to implant surfaces as a method of promotingintegration of the implant and inhibiting adverse membrane-mediated cellresponses of the host tissue has been studied.

U.S. Pat. No. 6,033,582 to Lee et al., which is incorporated byreference as if fully set forth herein, describes gas-plasma treatmentof implant device surfaces to obtain desirable surface features thatenhance and optimize adhesion of coating materials and/or tissueinteractions with the surface of a medical implant device.

An improvement in implant technology involves using implants as a sourceof drugs, agents, or other active substances, such as growth factors, toassist in wound healing and aid ingrowth of host tissue. Althoughcontrolled release of a therapeutic substance has received someinterest, these applications typically involve the immediate,unsustained release of the therapeutic substance.

SUMMARY OF THE INVENTION

An implant for use in biological/biomedical applications may be preparedby subjecting a substrate to a gas-plasma treatment. The substrate maybe a biocompatible material, including metals, ceramics, and polymers.More specifically, the substrate may be a bioresorbable polymer, such asa polylactide. The substrate may be a planar solid or a nonplanar solid.In some embodiments, the substrate may be a three-dimensional matrix. Inan embodiment, the implant is a medical implant.

Gas-plasma treatment of a substrate may include subjecting the substrateto a plasma formed by a reactive gas. A reactive gas may include oxygen.In an embodiment, a duration of the gas-plasma treatment may be fromabout 1 minute to less than about 5 minutes. During gas-plasmatreatment, the substrate may be exposed to a reactive gas at atemperature of less than about 50° C. In an embodiment, a substrate isexposed to a reactive gas at a pressure between about 0.01 and 10 torr.Energy supplied to a gas-plasma chamber may be between about 5 kJ andabout 10 kJ. A discharge frequency between about 10 kHz and about 100GHz may be used in the gas-plasma treatment. In an embodiment, thedischarge frequency may be between about 13 MHz and about 14 MHz.

In an embodiment, a substrate may be exposed to living cells. A portionof the living cells may become coupled to the substrate. Living cellsthat may be coupled to a substrate include endothelial cells, humanaortic endothelial cells, muscle cells, myocardial cells, and epithelialcells. Living cells coupled to a treated substrate may produce more of acellular product than living cells coupled to an untreated substrate.Cellular products that show an increase in production may includenucleic acids and proteins. More specifically, living cells coupled to atreated substrate may produce more of a growth factor than living cellscoupled to an untreated substrate. An increase in production may be seenfor growth factors including vascular endothelial growth factor, basicfibroblast growth factor, epidermal growth factor, andplatelet-endothelial cell adhesion molecule-1.

In an embodiment, an implant may be implanted into a person followinggas-plasma treatment of a substrate. Living cells coupled to the treatedsubstrate may produce more of a cellular product than living cellscoupled to an untreated substrate. In an embodiment, a substrate may beexposed to living cells prior to implanting the implant in a person.

BRIEF DESCRIPTION OF THE DRAWINGS

Other objects and advantages of the invention will become apparent uponreading the following detailed description and upon reference to theaccompanying drawings in which:

FIGS. 1A and 1B depict cross-sectional views of embodiments of ascaffold;

FIG. 2 is a histogram that shows VEGF absorbance for standard solutionsand for VEGF amounts released by HAECs coupled to treated and untreatedimplants in vitro; and

FIG. 3 shows graded blood vessel sprouting versus days in vivo fortreated and untreated implants.

While the invention is susceptible to various modifications andalternative forms, specific embodiments thereof are shown by way ofexample in the drawings and will herein be described in detail. Itshould be understood, however, that the drawings and detaileddescription thereto are not intended to limit the invention to theparticular form disclosed, but on the contrary, the intention is tocover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the present invention as defined by the appendedclaims.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Delivery of various chemical and biochemical factors to an implantationsite is used to enhance tissue regeneration in a vicinity of the site,promoting integration of an implant with host tissue. Delivery ofexogenous factors, however, introduces added complexity to theregeneration process. The method described herein enables the use ofsurface treatment of an implant to enhance the manufacture and releaseof cellular products by cells coupled to the implant. In this way,products synthesized by cells in situ may be used to regulate and toenhance tissue regeneration at an implantation site. Production anddelivery of factors in situ to specific host sites may modify the needfor delivery of exogenous factors.

Embodiments presented herein generally relate to a method of preparingan implant for use in biological/biomedical applications. The implant isformed from a substrate that is subjected to a gas-plasma treatment. Inan embodiment, the substrate may be exposed to living cells, such that aportion of the living cells are coupled to the treated substrate.Gas-plasma treatment of the substrate, according to the embodimentsdescribed herein, enhance the release of cellular products by cellscoupled to the substrate. As used herein, the term “implant” includesplanar and nonplanar solids having a regular or an irregular shape.Exposing the substrate to living cells may be accomplished by placingunattached living cells on the substrate or placing the substrate inmedia containing living cells. Alternatively, the substrate may beexposed to living cells when the implant is placed in contact with hosttissue. Living cells that are coupled to a substrate may be physicallyattached to or in physical contact with the substrate. Alternatively,living cells may be chemically coupled to a substrate. Living cells thatare chemically coupled to a substrate may exhibit cellular activity inresponse to properties of the substrate or in response to a cellinfluenced by the substrate.

Examples of medical implants include, but are not limited to, tissuescaffolds, bone implants, cartilage implants, implantable drug ormedication delivery systems, artificial skin, skin grafts, boneregeneration fillers, prostheses, and other attachable or implantableimplants designed to facilitate tissue or organ regeneration, repair,reconstruction, and/or growth. The term “implant” may also refer toarticles for other uses including, but not limited to, cell culturingand other biological and biomedical applications.

Implants may be composed of biocompatible materials including, but notlimited to, metals, ceramics, and polymers. An implant may besubstantially solid throughout, or at least a portion of an implant maybe porous and/or permeable. In an embodiment, an implant may be pre-castinto a desired shape to fit a specific application. In anotherembodiment, an implant may be cut or stamped from planar or nonplanarsolids, or the like, in shapes including, but not limited to, plates,disks, and cylindrical or conical plugs. In an embodiment, a shape of animplant may be modified as desired prior to gas-plasma treatment. Inanother embodiment, a shape of an implant may be modified as desiredfollowing gas-plasma treatment.

In biological/biomedical applications, implants may be advantageouslycomposed of biocompatible polymers. Bioresorbable polymers are a classof biocompatible polymers that are eroded by cellular action and/or arebiodegradable by action of non-living body fluid components.Bioresorbable polymers may be resorbed into host tissue over time.General examples of bioresorbable polymers include, but are not limitedto, polyesters, polyamides, polypeptides, polyfumarates,polysaccharides, polylactides, polyglycolides, polycaprolactones,polyanhydrides, polyamides, polyurethanes, polyesteramides,polyorthoesters, polydioxanones, polyacetals, polyketals,polycarbonates, polyorthocarbonates, polyphosphazenes,polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates,polyalkylene succinates, poly(malic acid), poly(amino acids),poly(methyl vinyl ether), poly(maleic anhydride), chitin, chitosan, andcopolymers, terpolymers, or higher poly-monomer polymers thereof orcombinations or mixtures thereof. Polymers that are degraded byhydrolysis may be advantageous.

Examples of bioresorbable polymers include poly(DL-lactic acid)(DL-PLA), poly(L-lactic acid) (L-PLA), poly(1-lactic acid) (P1LA),poly(glycolic acid) (PGA), poly(DL-lactic-co-glycolic acid) (PLG), andcombinations, mixtures or blends thereof. For PLG, the co-monomer(lactide:glycolide) ratios may be between about 100:0 and about 50:50lactic acid to glycolic acid. In an embodiment, the co-monomer ratiosare between about 85:15 and about 50:50 lactic acid to glycolic acid.Blends of PLA with PLG may also be used to prepare implants. Thedegradation products of these polymers are low molecular weightcompounds, such as lactic acid and glycolic acid, which enter intonormal metabolic pathways. Furthermore, copolymers of PLG offer theadvantage of a large range of degradation rates from a few days to a fewyears, depending on the copolymer ratio of lactic acid to glycolic acid.

In an embodiment, three-dimensional bioresorbable DL-PLA polymerscaffolds may be fabricated using a vibrating particle techniquedescribed in U.S. Pat. Nos. 6,187,329 and 6,255,359 to Agrawal et al.,both of which are incorporated by reference as if fully set forthherein. As used herein, the term “scaffold” means a three-dimensionalmatrix characterized by permeability and porosity that may be uniform ornonuniform. Scaffolds may be made of materials including, but notlimited to, tubular, fibrous, and woven polymers. Other startingmaterial configurations suitable for scaffold fabrication include wovenor knitted items, micro-or-nano-spheres (i.e., fullerenes), micro- ornano-tubes, cobweb-like configurations, or foams/sponge-like forms.FIGS. 1A and 1B depict cross-sectional views of embodiments of scaffold100. The circles in FIGS. 1A and 1B represent pores 102 in substrate104. FIG. 1A is a cross-sectional view of a substantially uniform,highly permeable and/or porous scaffold. FIG. 1B is a cross-sectionalview of a substantially uniform, highly permeable and/or porous scaffoldhaving different pore sizes.

To enhance biodegradation of polymers used in biological applications,an implant may also include one or more enzymes to facilitate thebiodegradation of the polymers used in the composition. Such enzymesinclude, but are not limited to, proteinase K, bromelaine, pronase E,cellulase, destranase, elastase, plasmin streptokinase, trypsin,chymotrypsin, papain, chymopapain, collagenase, chlostridopeptidase A,ficin, carboxypeptidase A, pectinase, pectinesterase, oxidoreductases,and oxidases. An appropriate amount of such a degradation enhancingagent may be used to regulate the lifetime of an implant. In variousembodiments, implants may further contain other materials such asfillers that may improve polymer strength, materials that may aid indegradation, materials that may retard degradation such as anti-oxidantsor anti-ozonants, biologically active agents, colorants, chromophores,light-activated (fluorescent or phosphorescent) tags, or any othermaterial that may alter or change the property of the compositions.

Bioactive agents may be added to implants to provide biological,physiological, or therapeutic effects. Such agents may be incorporatedinto implants before gas-plasma treatment or applied to implants aftergas-plasma treatment by dip or spray application or the like. Agents maybe chemically or ionically bonded to substrate sites. Agents applied tobioresorbable implants may be released during biodegradation of theimplant. Bioactive agents can be added to implants used as implants, forexample, to enhance cell growth and tissue regeneration, act for birthcontrol, or to cause nerve stimulation or bone growth.

Gas plasmas are created when energy sufficient to ionize atoms and/ormolecules in a reactive gas causes ionization and subsequent generationof free electrons, photons, free radicals, and ionic species. Unlikedistinct phase changes in other states of matter, transition of a gas orvapor from an unexcited, electrically stable state to an ionized plasmastate generally occurs through a continuous process. The excitationenergy supplied to a gas to form a plasma can originate from electricaldischarges, direct currents, radio frequencies, microwaves, or otherforms of electromagnetic radiation.

Gas-plasma techniques for modifying the surface characteristics of manymaterials are known. Specific applications for surface modifiedmaterials have been described for both microcircuit and medical implantdevice technology. In the medical implant industry, the use of plasmatreatment of materials has generally focused on surface conditioning.U.S. Pat. Nos. 3,814,983; 4,929,319; 4,948,628; 5,055,316; 5,080,924;5,084,151; 5,217,743; 5,246,451; 5,260,093; 5,262,097; 5,364,662;5,451,428; 5,476,509; 5,229,172; 5,236,563; and 5,543,019, all of whichare incorporated by reference as if fully set forth herein, describesurface conditioning of medical implants and other devices by radiofrequency (RF) gas plasmas.

Many gas-plasma treatment techniques use RF radiation to break surfacepolymer bonds. This process generates ions and free radicals, setting upfavorable conditions for subsequent RF plasma-induced polymerization andgrafting of monomers to a substrate as described in U.S. Pat. No.5,080,924, which is incorporated by reference as if fully set forthherein. In another application, similar covalent bonding of polymericbiocompatible materials to intraocular lenses via RF plasma grafting wasachieved, creating a microscopically smooth surface as described in U.S.Pat. No. 5,260,093, which is incorporated by reference as if fully setforth herein.

Gas-plasma treatment may take place in a chamber capable of sustaining aplasma at low pressures. A substrate in the chamber is exposed to theplasma. In an embodiment, a RF plasma may operate at a frequency betweenabout 10 kHZ and about 100 GHz, between about 1 MHz and about 100 MHz,or between about 13 MHz and about 14 MHz. Inert and/or reactive gasesmay be used in gas-plasma treatment. Examples of inert gases include,but are not limited to, noble gases such as helium and argon. Examplesof reactive gases include, but are not limited to, oxygen, water vapor,hydrogen peroxide vapor, hydrogen, nitrogen, ammonia, and mixturesthereof. In an embodiment, an internal pressure during the gas-plasmatreatment may be from about 10⁻⁴ torr to about 100 torr, from about 0.01torr to about 10 torr, or about 0.15 torr. In an embodiment, atemperature inside the plasma chamber may be less than about 50° C.,between about 10° C. and about 40° C., or about 25° C.

Duration of a gas-plasma treatment, together with RF power, maydetermine the energy delivered to the plasma chamber during treatment.In an embodiment, duration of the gas-plasma treatment may be greaterthan about 1 minute to less than about 5 minutes, between about 2minutes and about 4 minutes, or about 3 minutes. In an embodiment, theRF power may range between about 25 watts and about 250 watts, betweenabout 40 watts and about 100 watts, or about 60 watts. In someembodiments, treatment duration and RF power may be selected such thatthe product of power in watts and treatment duration in seconds is equalto a supplied energy between about 2 kJ and about 20 kJ, between about 5kJ and about 10 kJ, or about 7.5 kJ. For example, a three-minutetreatment with a RF power of 100 watts delivers a supplied energy of100 watts×180 seconds=18,000 J,or 18 kJ, to the plasma chamber during treatment. In some embodiments,the RF power and/or phase may be varied during treatment.

U.S. Pat. No. 6,033,582 to Lee et al. describes a reactive plasmaetching process that modifies the surface of an implant such that theresulting roughness, porosity, and texture are optimized for applicationof a coating. Biodegradable polymers such as poly(lactic acid) andpoly(glycolic) acid are etched with noble gas (e.g., helium or argon) RFplasma in the presence of a reactive gas such as water vapor, oxygen, orhydrogen. The resulting roughened surface is shown to promote cellularingrowth and allow improved adhesion of bioactive coatings, such asproteins, antibiotics, and nucleic acids, typically chosen to promoteappropriate physiological responses in a host. It is further describedthat textured surfaces may be conditioned for cell attachment throughapplication of growth matrix materials or components. Suitableconditioning materials are applied through dip coating and drying,followed by application of cells either in vitro or in situ. A post-etchapplication of a growth-promoting interface such as collagen orpoly-lysine is proposed to promote adhesion of endothelial cells.

Gas-plasma treatment may be applied, for example, to implants fordiseased or impaired organs or implants used to grow whole, artificialorgans. Starting materials for these implants or artificial organs aregenerally biocompatible and may be bioresorbable. Starting materialsshould provide appropriate structural integrity and support and shouldbe able to withstand selected gas-plasma conditions. Once a substrate istreated, appropriate cell growth materials and processes may be appliedto the implant.

Gas-plasma treatment may induce formation of free radicals on asubstrate. In an embodiment, gas-plasma treatment with a reactive gasthat includes oxygen may induce formation of oxide free radicals on asubstrate. As shown below, parameters chosen for a gas-plasma treatmentmay influence relative density of free radicals formed on the substrate.

Electron spectroscopy for chemical analysis (“ESCA”) data fromthree-dimensional plug-shaped DL-PLA implants (5 mm diameter×2 mm thick)were used to compare mass concentration percentages corresponding tocarbon atoms in various bonding configurations, including C═O, C—O, C—C,and C—O—O bonds, where applicable. Table 1 shows the percentages of C═O,C—O, and C—C bonds in an untreated substrate. Tables 2-4 show data fromsubstrates subjected to gas-plasma treatment with an RF setting of 100watts, and RF frequency of 10-11 MHz, a vacuum of 100-200 mtorr., usingoxygen as the reactive gas. Table 2 shows percentages of C═O, C—O, C—C,and C—O—O bonds on a substrate following a 1 minute gas-plasma treatmentwith a RF power of 100 watts (supplied energy=6 kJ). Table 3 showspercentages of C═O, C—O, C—C, and C—O—O bonds on a substrate following a3 minute gas-plasma treatment with a RF power of 40 watts (suppliedenergy=7.2 kJ). Table 4 shows percentages of C═O, C—O, C—C, and C—O—Obonds on a substrate following a 3 minute gas-plasma treatment with a RFpower of 100 watts (supplied energy=18 kJ).

TABLE 1 Untreated Peak Position (eV) Mass Concentration % C═O 287.02033.35 C—O 284.919 33.33 C—C 282.946 33.32

TABLE 2 Gas-plasma treated: 1 minute, 100 watts Peak Position (eV) MassConcentration % C═O 282.843 31.47 C—O 284.748 34.59 C—C 286.851 30.45C—O—O 287.800 3.49

TABLE 3 Gas-plasma treated: 3 minutes, 40 watts Peak Position (eV) MassConcentration % C═O 286.941 39.12 C—C 284.802 31.77 C—C 282.907 22.70C—O—O 288.797 6.41

TABLE 4 Gas-plasma treated 3 minutes, 100 watts Peak Position (eV) MassConcentration % C═O 282.847 37.88 C—O 284.712 32.48 C—C 286.946 29.64

Data from the untreated substrate in Table 1 suggest that C═O, C—O, andC—C bonds are present in approximately equal numbers, with each bondtype corresponding to approximately one-third of the carbon-containingsurface bonds. Data from Table 2 for a substrate treated at a RF powerof 100 watts for 1 minute suggest that gas-plasma treatment induced theformation of oxide radicals, with relative occurrences of C═O, C—O, C—C,and C—O—O bonds measured as 31.47%, 34.59%, 30.45%, and 3.49%,respectively. Data from Table 3 for a substrate treated at a RF power of40 watts for 3 minutes suggest that gas-plasma treatment induced an evengreater formation of oxide radicals, with relative occurrences of C═O,C—O, C—C, and C—O—O bonds measured as 39.12%, 31.77%, 22.70%, and 6.41%,respectively. Thus, the relative percentage of oxide radicals is almosttwice as high for a 40 watt, 3 minute gas-plasma treatment than for a100 watt, 1 minute gas-plasma treatment. Data from Table 4, withrelative occurrences of C═O, C—O, and C—C bonds measured as 31.47%,34.59%, and 30.45%, respectively, suggest that treatment at a RF powerof 100 watts for 3 minutes does not promote formation of oxide radicals.

Gas-plasma treatment may cause a hydrophobic substrate to become morehydrophilic. As used herein, the term “hydrophilic” means having atendency to bind water. The term “hydrophobic” is used herein to meanhaving a tendency to repel water. A more hydrophilic substrate maypromote cellular adhesion and proliferation. In an embodiment, agas-plasma treated substrate may be exposed to living cells, such that aportion of the living cells become coupled to the substrate. Examples ofcells that may be coupled to treated substrates include, but are notlimited to, endothelial cells, muscle cells, myocardial cells, andepithelial cells. In an embodiment, the endothelial cells may be humanaortic endothelial cells (HAECs).

Living cells coupled to a gas-plasma treated substrate may produce andrelease more of a cellular product than cells coupled to an untreatedsubstrate. Such cellular products include, but are not limited to,proteins and nucleic acids. In some embodiments, living cells coupled toa gas-plasma treated substrate produce and release more β-tubulin thancells coupled to an untreated substrate. β-tubulin is a protein that maycorrelate with cell proliferation as well as increased expression ofother cellular products, such as growth factors. Examples of growthfactors include, but are not limited to, colony stimulating factor,epidermal growth factor (EGF), erthyropoietin, fibroblast growth factor,neural growth factor, human growth hormone (HGH), platelet derivedgrowth factor (PDGF), insulin-like growth factor (ILGF), fibronectin(FN), endothelial cell growth factor (ECGF), vascular endothelial growthfactor (VEGF), cementum attachment extracts (CAE), basic fibroblastgrowth factor (bFGF), periodontal ligament cell growth factor (PDGF),epidermal growth factor (EGF), protein growth factor interleukin-1(IL-1), transforming growth factor (TGF beta-2), human alpha thrombin(HAT), osteoinductive factor (OIF), and bone morphogenic protein (BMP),and platelet-endothelial cell adhesion molecule-1 (PECAM-1).

In an embodiment, living cells coupled to a gas-plasma treated substratemay produce more VEGF than living cells coupled to an untreatedsubstrate. VEGF regulates endothelial cell growth and new blood vesselformation (angiogenesis). VEGF is capable of influencing the expressionof other cellular products.

FIG. 2 is a histogram that shows mean absorbance (540 nm) ±standarddeviations for VEGF standards 110 ranging from 15.6 to 125.0 pg/mL.Absorbance measurements for the VEGF standards were used to quantify anamount of VEGF released into the media from an in vitro study of HAECsincubated for 10 days near and within untreated and oxygen gas-plasmatreated three-dimensional DL-PLA implants. VEGF levels released by HAECsinto the media were higher (statistical significance p=0.016) for thetreated implants 112 (range: 15.6-34 pg/mL) than for the untreatedimplants 114(range: ≦15.6 pg/mL). After 18 days, HAECs within treatedimplants expressed higher VEGF intensities. Experimental VEGF quantitieswere determined using an enzyme-linked immunosorbant assay (“ELISA”: R&DSystems; Minneapolis, Minn.).

In an embodiment, living cells coupled to a gas-plasma treated substratemay produce more PECAM-1 than living cells coupled to an untreatedsubstrate of the same initial composition as the treated substrate.PECAM-1 is a growth factor influenced by VEGF that promotes cellularmigration, survival, and replication.

Levels of PECAM-1 were investigated qualitatively in the in vitro studydocumented in FIG. 2. By 18 days, PECAM-1 within HAECs incubated withingas-plasma treated implants was heavily expressed throughout thecytoplasm and along the cytoskeleton of closely aligned andinterconnecting cells. Migrating HAECs in untreated implants exhibitedinferior and diffuse cytoplasmic PECAM-1 expression.

In an embodiment, free radicals on implant surfaces may simulate in vivovascular injury, thereby promoting endothelial cell activation andinduction of blood vessel formation, or angiogenesis. Angiogenesis wasdocumented in an in vivo study of oxygen gas-plasma treatedthree-dimensional DL-PLA scaffolds above the omentum of nude mice. (Theomentum is a fold of membrane extending from the stomach to adjacentorgans in the abdominal cavity.) Visible vessels (>500 μm) were scoredbased on distance from the implants by two blinded observers. Thegrading system for the development of new vessels was:

-   -   Grade 1: Vessels seen on the implant.    -   Grade 2: Vessels seen on the implant and outside the implant (<5        mm).    -   Grade 3: Vessels seen on the implant and outside the implant        (5-10 mm).    -   Grade 4: Vessels seen on the implant and outside the implant        (>10 mm).        FIG. 3 shows mean graded blood vessel sprouting versus days        ±standard deviation in vivo for untreated implants not exposed        to HAECs 120, untreated implants exposed to HAECs 122, treated        implants not exposed to HAECs 124, and treated implants exposed        to HAECs 126.

An initial angiogenic response, or development of blood vessels, isnoted for all the implants. More extensive blood vessel development isseen in the case of treated implants. The omentum and/or the skin of themice with treated implants revealed angiogenesis that started early (12days) and persisted throughout the experiment (72 days). There was aprogressive increase in the grading between days 12 and 72 in thetreated implants exposed to HAECs 126 and a steady high scoring in thetreated implants not exposed to HAECs 124. The untreated implant groupsshowed less vessel formation with a maximum average grade of 1.5 for theuntreated implants exposed to HAECs 122 and a maximum average grade of1.2 for the untreated implants not exposed to HAECs 120.

A statistically significant difference is seen between the mice in thetreated groups and untreated groups at all measured intervals (12, 18,24, and 72 days). The difference is most noticeable between the micewith treated implants exposed to HAECs 126 and those with untreatedimplants not exposed to HAECs 120. The mice with untreated implantsexposed to HAECs 122 showed slightly enhanced angiogenesis compared tothe untreated implants not exposed to HAECs 120. The increase in thesize of the vessels for the untreated implants exposed to HAECs 122relative to the untreated implants not exposed to HAECs 120, however,was not statistically significant. The increase in blood vesselsprouting over time for treated implants exposed to HAECs 126 isstatistically significant, as indicated by p values ranging from 0.029to 0.002.

In the same study, VEGF expression at day 18 was more evident in thevessels in and around the implants in mice with treated implants than inmice with untreated implants. PECAM-1 expression was evaluatedqualitatively at day 18 in the omentum of mice with treated anduntreated implants. A substantial proportion of the mice with treatedimplants showed evidence of PECAM-1 expression in tissues surroundingthe implants. Untreated implants revealed less intense PECAM-1expression.

As a bioresorbable implant, such as a DL-PLA implant, begins to degradewithin a host, an injury stimulus of substrate free radicals maydiminish. As a result, the production of angiogenic growth factors suchas VEGF may taper off. This internal control may prevent the formationof vascular tumors. In addition, the local release of growth factors inthe vicinity of the implant may reduce the need for delivery ofexogenous growth factors.

EXAMPLE 1

The following example describes coupling of HAECs to L-PLA films invitro.

Culture: HAECs (Clonetics 1998, Passages 5-8: Cambrex Corporation; EastRutherford, N.J.) were seeded using basal MCDB-131 (Sigma; St. Louis,Mo.) media. The chemically defined MCDB-131 was supplemented with 1μg/mL hydrocortisone (HC: Sigma), 250 ng/mL basic fibroblast growthfactor (bFGF: PeproTech; Rocky Hill, N.J.), 1 μg/mL epidermal growthfactor (EGF: Gibco; Rockville, Md.), 100 I.U. penicillin/streptomycin(P/S; Cellgro: Herndon, Va.), and 10% iron-supplemented bovine calfserum (BCS; Hyclone: Logan, Utah). The supplemented MCDB-131 media wasbuffered with sodium bicarbonate and HEPES, free acid. The media had anaverage beginning pH of 7.35 and an osmolarity of 324 mOsm at 22° C.Prior to seeding, HAECs were treated with 0.5% trypsin plus 0.53 mM EDTA(Gibco). HAECs were collected by centrifugation, re-suspended insupplemented MCDB-131, and then seeded onto the center of bioresorbablepolymer films, prepared as described below. The endothelial cultureswere grown in a 37° C. humidified 5% CO₂/95% O₂ incubator. Coupled HAECswere fed every two to three days with supplemented MCDB-131 media.

Bioresorbable polymer films: L-PLA films were prepared by dissolving thedry L-PLA (MW 450 kD; Birmingham Polymers, Inc.: Birmingham, Ala.) inmethylene chloride. The dissolved L-PLA was poured onto a glass mold. Aglass recrystallization dish was placed over the solution in the mold tocontrol airflow and evaporation rate at 4° C. After the solventevaporated, the films were rinsed with sterile distilled water andremoved from the glass plates. 15 mm-diameter circles were extractedfrom the L-PLA films using a metal punch. Gas-plasma treatment wasperformed using the STERRAD sterilization technology (AdvancedSterilization Products; Irvine, Calif.), which involved the combined useof hydrogen peroxide and low-temperature gas plasma. The processtemperature did not exceed 50° C. and treatment occurred in a lowmoisture environment.

The treated films showed greater endothelial proliferation thanuntreated films, as indicated by increased β-tubulin expression.

EXAMPLE 2

The following example describes coupling of HAECs to three-dimensionalbioresorbable DL-PLA implants in vitro.

Culture: HAECs (Passages 4-7) were seeded at sub-confluent densitiesusing basal MCDB-131 (Sigma) media supplemented with 1 μg/mLhydrocortisone (HC: Sigma), 250 ng/mL basic fibroblast growth factor(bFGF: PeproTech), 1 μg/mL epidermal growth factor (EGF: Gibco), 100I.U. penicillin/streptomycin (P/S: Cellgro), and 10% iron-supplementedbovine calf serum (BCS; Hyclone) buffered with sodium bicarbonate andHEPES, free acid (pH 7.35; 324 mOsm). HAECs were treated with 0.5%trypsin plus 0.53 mM EDTA (Gibco), collected by centrifugation,re-suspended in supplemented MCDB-131, and then seeded onto the centerof bioresorbable DL-PLA implants housed in 96-well plates (Corning;Corning, N.Y.). When seeded onto implants made as described below, themedia was switched to RPMI 1640 (Cellgro) with the same supplements and10% BCS (pH 7.35; 280 mOsm). Endothelial cultures were grown in a 37° C.humidified 5% CO₂/95% O₂ incubator (VWR; West Chester, Pa.). HAECs werefed every two to three days during the three to eighteen days ofincubation.

Bioresorbable polymer implants: Using sterile techniques,three-dimensional substrates were fabricated using DL-PLA with aninherent viscosity of 0.63 dL/g (Birmingham Polymers, Inc.). Implantswere fabricated using a vibrating particle technique described in U.S.Pat. Nos. 6,187,329 and 6,255,359 to Agrawal et al., resulting in highlyporous and permeable plug-shaped implants (5 mm diameter×2 mm thick).Implants were treated using a RF (13.18 MHz) glow discharge of oxygen ina 2 ¾ inch glass chamber (Harrick Plasma Cleaner: Harrick ScientificCorporation; Ossining, N.Y.). The internal pressure was 0.15 torr. Theprocess temperature did not exceed 50° C., and treatment occurred in alow-moisture environment.

VEGF expression was greater in the wells of treated implants than thewells of untreated implants. VEGF levels outside the treated implantswere also elevated. PECAM-1 within HAECs incubated within gas-plasmatreated implants was heavily expressed throughout the cytoplasm andalong the cytoskeleton of closely aligned and interconnecting cells.Migrating HAECs in untreated implants exhibited inferior and diffusecytoplasmic PECAM-1 expression.

EXAMPLE 3

The following example describes coupling of HAECs to three-dimensionalDL-PLA implants for use as implants in nude mice.

Three-dimensional scaffolds were fabricated using a DL-PLA polymer withan inherent viscosity of 0.63 dl/g (Birmingham Polymers, Inc.).Scaffolds were fabricated using a vibrating particle technique describedin U.S. Pat. Nos. 6,187,329 and 6,255,359 to Agrawal et al., resultingin highly porous and permeable scaffolds. Implanted scaffolds (5 mmdiameter×2 mm thick) were cut from the composite using a punch, immersedin water for 48 hours to remove the NaCl, and dried in a vacuum. Allmaterials and instruments used for the scaffold fabrication weresterilized beforehand.

Implants were gas-plasma treated using a RF glow discharge of oxygen(Harrick Plasma Cleaner) in a 2¾ inch glass chamber at a frequency of13.18 MHz, at a power of 100 Watts for 3 minutes, and at an internalpressure of 0.15 torr. The process temperature did not exceed 50° C. andtreatment occurred in a low-moisture environment.

3×10⁴ HAECs (Passages 5-8, BioWhittaker: Walkersville, Md.) were allowedto attach to each free floating implant using RPMI 1640 supplementedwith 1 μg/mL hydrocortisone (Sigma), 250 ng/mL bFGF (PeproTech), 1 μg/mLepidermal growth factor (Gibco); 100 I.U. penicillin/streptomycin(Cellgro) and 10% BCS (Hyclone). Media was replaced every 2-3 days. 2-4hours prior to implantation, near confluent 6-day endothelializedimplants were switched to media supplemented as listed above withoutserum.

Results: Formation of new vessels was seen in abdominal skin when micewere alive. Formation of new vessels was seen in the peritoneum,abdominal skin, or both postmortem. Mice in each group showedimplant-induced angiogenesis. The angiogenic response was morenoticeable, however, in mice with treated implants. VEGF and PECAM-1expression were greater in and around treated implants. β-tubulinexpression was enhanced in HAECs located in the treated implants.

EXAMPLE 4

The following example describes coupling of HAECs to three-dimensionalDL-PLA implants used as implants in a nude mouse omental model.

Culture: HAECs (BioWhittaker) were seeded with 3×10⁴ HAECs using RPMI1640 supplemented with 1 mg/mL hydrocortisone (HC: Sigma), 250 ug/mLbasic fibroblast growth factor (bFGF: PeproTech), 1 ug/mL epidermalgrowth factor (EGF: Gibco), 100 I.U. penicillin/streptomycin (P/S:Cellgro), and 10% iron-supplemented bovine calf serum (BCS: Hyclone).Supplemented RPMI 1640 was replaced every 2-3 days.

Bioresorbable polymer implant: Three-dimensional scaffolds werefabricated using DL-PLA with an inherent viscosity of 0.63 dl/g(Birmingham Polymers, Inc.). Scaffolds were fabricated using a vibratingparticle technique described in U.S. Pat. Nos. 6,187,329 and 6,255,359to Agrawal et al., resulting in highly porous and permeable scaffolds.Briefly, 0.35 gm of DL-PLA was dissolved in 3.25 ml of pure acetoneunder continuous stirring. Approximately 2.25 gm of sodium chloride(NaCl) particles (250 μm≦size≦500 mm) were spread evenly at the bottomof a Teflon™ mold with a rectangular well cavity (33×20×15 mm). Next,the polymer solution was poured onto the salt in an even fashion. Themold was then placed under continuous airflow conditions and vibratedusing a Thermolyne Maxi Mix II (Barnstead/Thermolyne; Dubuque, IA) for 8minutes. An additional 4.5 gm of salt were added during this period. Themold was continuously vibrated until the solvent evaporated, leavingbehind a solid salt-polymer composite. This composite was placed in aheated vacuum at 45° C. and 5 torr for 24 hours. Disk-shaped scaffolds(5 mm diameter×2 mm thick) were cut from the composite using a punch,immersed in water for 48 hours to remove the NaCl, and dried in avacuum. All materials and instruments used for the scaffold fabricationwere sterilized beforehand.

Implants were gas-plasma treated using a RF glow discharge of oxygen(Harrick Plasma Cleaner) in a 2¾ inch glass chamber at a frequency of13.18 MHz, at a power of 100 Watts for 3 minutes, and at an internalpressure of 0.15 torr. The process temperature did not exceed 50° C. andtreatment occurred in a low-moisture environment.

Results: Qualitative analysis of implants revealed enhanced localexpression of VEGF and PECAM-1 in and around the treated implants. Therewas also a more organized local new blood vessel sprouting in thevicinity of treated implants.

1. An implant prepared by a process comprising: subjecting abioresorbable polymeric substrate to a gas-plasma treatment, wherein thebioresorbable polymeric substrate comprises a polylactide polymericmaterial, wherein subjecting the substrate to the gas-plasma treatmentcomprises exposing the substrate to a reactive gas, wherein the reactivegas comprises oxygen, and wherein the supplied energy during thegas-plasma treatment is between about 5 kJ and about 10 kJ at atemperature of less than about 50 C, a pressure between about 0.01 torrand about 10 torr, and a discharge frequency between about 13 MHz andabout 14 MHz, wherein the reactive gas comprises an oxygen contentsufficient to provide oxide free radicals on the substrate when thesubstrate is subjected to the gas-plasma treatment; and exposing thesubstrate subjected to the gas-plasma treatment to living cells thatproduce vascular endothelial growth factor (VEGF), wherein a portion ofthe living cells that produce VEGF become coupled to the substrate; andwherein the living cells that produce VEGF coupled to the substratesubjected to the gas-plasma treatment produce more VEGF than livingcells that produce VEGF when coupled to the substrate not subjected tothe gas-plasma treatment.
 2. A method of preparing an implant,comprising: subjecting a bioresorbable polymeric substrate to agas-plasma treatment, wherein the bioresorbable polymeric substratecomprises a polylactide polymeric material, wherein subjecting thesubstrate to the gas-plasma treatment comprises exposing the substrateto a reactive gas, wherein the reactive gas comprises oxygen, andwherein the supplied energy during the gas-plasma treatment is betweenabout 5 kJ and about 10 kJ at a temperature of less than about 50 C, apressure between about 0.01 torr and about 10 torr, and a dischargefrequency between about 13 MHz and about 14 MHz, wherein the reactivegas comprises an oxygen content sufficient to provide oxide freeradicals on the substrate when the substrate is subjected to thegas-plasma treatment; and exposing the substrate subjected to thegas-plasma treatment to living cells that produce vascular endothelialgrowth factor (VEGF), wherein a portion of the living cells that produceVEGF become coupled to the substrate; and wherein the living cells thatproduce VEGF coupled to the substrate subjected to the gas-plasmatreatment produce more VEGF than the living cells that produce VEGF whencoupled to the substrate not subjected to the gas-plasma treatment. 3.The method of claim 2, wherein the substrate comprises athree-dimensional matrix.
 4. The method of claim 2, wherein thesubstrate comprises a planar solid.
 5. The method of claim 2, whereinthe substrate comprises a nonplanar solid.
 6. The method of claim 2,wherein the implant is a medical implant.
 7. The method of claim 2,wherein the reactive gas consists essentially of oxygen.
 8. The methodof claim 2, wherein the living cells comprise endothelial cells.
 9. Themethod of claim 2, wherein the living cells comprise human aorticendothelial cells.
 10. The method of claim 2, wherein the living cellscomprise muscle cells.
 11. The method of claim 2, wherein the livingcells comprise myocardial cells.
 12. The method of claim 2, wherein theliving cells comprise epithelial cells.